Identification of Potent Bioassay Guided Terpenoid and Glycoside Root Fractions of Astragalus candolleanus against Clinically Significant Bacterial Strains

Antibiotic resistance represents one of the biggest challenges, and there is an urgent need for plant-based antimicrobial agents that enable managing this crisis effectively. In this work, we aimed to investigate the antibacterial activity of Astragalus candolleanus (A. candolleanus) hydromethanolic root extract against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Kocuria rhizophila) strains by the cup-plate method. The root was powdered and extracted with 70% methanol by cold maceration for 5 days. Preliminary phytochemical screening was performed with different solvents in the order of increasing polarity. Pure compounds were isolated by column chromatography and were characterized through liquid chromatography-mass spectrometry. Targeted predictions of the isolated compounds were also studied using Swiss Target prediction software and prediction of activity spectra for substances. The extract showed a broad zone of inhibition against pathogenic bacteria. Four pure compounds were isolated, of which a novel terpenoid compound has been identified as stemmadenine along with scillirosidin, cephalotaxine, and myxoxanthophyll. The structures of the isolated phytoconstituents were elucidated by spectral analysis. The four pure components isolated from the roots of A. candolleanus are suggested to be effective against tested pathogens. Overall results of drug design suggest that myxoxanthophyll is a promising bioactive compound endowed with antibacterial activity.


Introduction
Antibiotic resistance represents a serious problem for public health [1,2]. Despite the enormous efforts to limit this phenomenon, an increasing number of antimicrobials, which were designed to kill or arrest the growth of bacteria, viruses, or fungi, are becoming ineffective, so antibioticresistance-related therapeutic failure is currently a real emergence worldwide [3][4][5][6].
is condition significantly affects our ability to prevent and treat infectious diseases promptly. In the last few decades, the discovery and development of novel anti-infective drugs have represented an active research area. Concerning this, natural compounds have historically been recognized as a rich source of antiinfective drugs, which provided penicillin in 1940, tetracyclines in 1948, and glycopeptides in 1955 [7]. is evidence promoted the study of natural products, considering a valid source of bioactive molecules that could help to manage this crisis effectively. Astragalus rhizanthus subsp. Candolleanus Benth.
(synonym Rudravanti) (A. candolleanus) belong to the family Fabaceae, and it is a wild-growing herb widely spread in the Himalayas from Jammu & Kashmir to Uttarakhand provinces in India [8,9]. A. candolleanus is endowed with several health benefits, such as immune-boosting, antiaging, and anti-inflammatory effects [10,11]. is plant has also been useful in the treatment of blood and skin diseases, tuberculosis, and joint pains and as an antidiabetic medication [12][13][14][15]. A few of the most common bacteria that can cause complicated life-threatening infections like septicemia are Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), and Kocuria rhizophila (K. rhizophila) [16]. Interestingly, a previous study showed the antibacterial and antibiofilm activities of Astragalus angulosus ethanolic extract against three Gram-positive strains (Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis) and two Gram-negative strains (E. coli and P. aeruginosa) [17][18][19]. Albayrak and Kaya investigated the antibacterial and antifungal activities of four Astragalus species (Astragalus gummifer, Astragalus microcephalus, Astragalus talasseus, and Astragalus acmophyllus) endemic to Turkish flora. Specifically, they performed the disk diffusion assay against P. aeruginosa (low activity with respect to standard antibiotics tetracycline and oxacillin) and Candida albicans (no activity) [20]. Recently, Guo et al. demonstrated the antibacterial activity of Astragalus membranaceus ethyl acetate aerial parts extract. In particular, it was effective against the Gram-positive strain B. subtilis [21]. is activity could be related to the high concentration of flavonoids in the extract [22]. However, to date, only a few studies focused on the biological activity and chemical composition of A. candolleanus root extracts. Considering that literature data reported outstanding biological activity of the root extracts from other Astragalus species, we proposed to fill the knowledge gap on A. candolleanus by investigating the chemical profile and antimicrobial activity of its extract. After a thorough literature survey on Astragalus species with emphasis on its phytochemical screening in various parts of the plant (root, leaf, and fruit), the root extract was considerably found to be enriched with phytoconstituents [23][24][25][26]. Interestingly, it has been found that the root of Astragalus mongholicus is colonized by several bacterial species, which are able to modify the secondary metabolites of the plant. According to such a study, the biological properties and chemical features of a phytoextract can be the results of the interaction between bacteria and plants [27]. So, the present work aimed to investigate the antibacterial potential of A. candolleanus root extract against the most common bacteria involved in septicemia and, subsequently, identify the active phytoconstituents responsible for the biological activity. For this purpose, by column chromatography, the root extract was fractionated to isolate the various pure phytoconstituents. ese pure components were elucidated using the mass spectrometry technique.

Preparation of Plant Extract.
A. candolleanus roots were dried at room temperature and reduced to a coarse powder. About 20 g of the powdered root was extracted with 200 mL of methanol-water mixture (7 : 3 v/v) by cold maceration for 5 days. Afterwards, the mixture was decanted and filtered to get the crude extract. e extract was then concentrated under reduced pressure through a rotavapor (Buchi-R100), followed by drying on a desiccator.

Antibacterial Activity.
e bacterial strains were purchased from the American Type Culture Collection Centre (ATCC) through an authorized vendor, Hi-Media Pvt. Ltd. Resources (reagents and apparatus) from the Indian Pharmacopoeia Commission, Ghaziabad, and used to carry out this research. e antibacterial activity of hydro-methanolic root extract of A. candolleanus was evaluated by the cupplate method against B. subtilis (ATCC 6633), E. coli (ATCC 9637), K. pneumoniae (ATCC 10031), K. rhizophila (ATCC 9341) P. aeruginosa (ATCC 25619), Salmonella typhimurium (ATCC 1428), and S. aureus (ATCC 6538). Bacteria were cultured on nutrient agar media (Hi-media). e method of Ali et al. with some modifications was used for the antibacterial assay [19]. e 5 mm bores were made in the agar medium through sterile cork borers. 100 μL of extract (100 mg/mL) in different dilutions (from 5 to 80 μg/ml) was placed in the wells along with DMSO as a negative control.
DMSO was diluted in a 1 : 100 ratio and did not affect bacterial growth. e activity of the natural extract was compared to that of the standard antibiotic gentamycin, in concentrations ranging from 5 to 40 µg/ml. e agar plates were incubated for 24 h at 30-35°C. e parameters used for observation were the estimation of the inhibition zone of bacterial growth surrounding the wells. e unit for the diameter of the inhibition zone was taken in millimetres (mm).

Isolation of Constituents from A. candolleanus Roots.
A column of 400 mm length with 500 mL reservoir capacity, 30 mm internal diameter, and 40 mm outer diameter was prepared with the wet packing method using silica (100-200 mesh size) as a stationary phase. e hydro-methanolic root extract was packed into the column and was eluted in a sequence from nonpolar solvents to polar solvents to obtain different pure fractions. e similarity profile of fractions was checked by thin layer chromatography, and similar fractions were identified. Pure components could be obtained in the case of root extract eluted with 70% v/v ethanolwater. e isolated and purified compounds were analyzed using Fourier-transform infrared spectroscopy (Shimadzu, IR Affinity-1) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) (Agilent 6520).

Determination of Melting Point of the Isolated Compounds.
e melting point is an intensive physical property that is characteristic of a specific compound. us, the melting points of the isolated compounds were determined to ensure their purity. All melting points were measured on a melting point apparatus (Accuma India Digital Melting/Boiling point apparatus).

Results and Discussion
After evaporation of the solvent, the screening for active phytoconstituents in the semisolid hydro-methanolic root extract of A. candolleanus showed the presence of alkaloids, glycosides, terpenes, lipids, volatile oil, gums, and mucilage. Table 1 demonstrates the results of the phytochemical analysis: a pilot screening was performed using several solvents/solvent systems characterized by a different polarity.
e hydro-methanolic root extract of A. candolleanus was able to inhibit the growth of all tested Gram-positive and Gram-negative strains. Specifically, as reported in Table 2 and Figure 1, the extract exerted the antibacterial activity already at the concentration of 5 µg/mL; however, at the concentration of 10 µg/mL, it produced a broader inhibition zone against S. aureus (28.6 mm), S. typhimurium (25.5 mm), K. rhizophila (24.6 mm), K. pneumoniae (21.8 mm), and E. coli (22.7 mm). Besides, at this concentration, A. candolleanus extract showed a strong growth inhibition for B. subtilis (41.2 mm) and P. aeruginosa (41.6 mm). ese bacteria are seen to be mainly responsible for recurrent bacterial infections. e obtained results indicated that the hydro-methanolic root extract of A. candolleanus can be considered a promising antimicrobial agent, due to its broad zone of inhibition against pathogenic bacteria. Our results revealed that the extract was more efficient in counteracting the growth of Gram-positive strains compared to the Gram-negative ones, except for P. aeruginosa. In this regard, it is worth noting that Gramnegative bacteria are generally more resistant to the natural antimicrobial agents compared to the Gram-positive ones.
is condition reflects the different composition of the cell wall between the two types of bacteria [32]. e bacterial cell wall is a multilayered structure that protects microorganisms from different environmental conditions and antimicrobial stress. Besides, it confers a characteristic shape and prevents cell rupture. In particular, the Gram-positive bacterial cell wall is formed by a thick layer of peptidoglycan, which is cross-linked with long anionic polymers called teichoic acids. Conversely, Gram-negative bacteria are endowed with a thinner layer of peptidoglycan, surrounded by an outer membrane containing lipopolysaccharides, extremely selective to the passage of xenobiotics [33,34]. is structural organization constitutes an efficient barrier against external agents, making the Gram-negative related infections very difficult to treat [35]. Concerning the molecular aspect, there are numerous mechanisms of action through which antimicrobial agents act, including inhibition of synthesis of bacterial proteins, inhibition of cell wall synthesis, damage to the bacterial cell membrane, interference with DNA replication/repair, and their metabolic pathway [36]. e characterization of phytoconstituents from the roots of A. candolleanus allows the identification of pure compounds. e isolated and purified compounds were analyzed using FTIR and LC-MS-MS techniques. e results and the inference of the characterization of A. candolleanus root by FTIR are presented in Table 3. e melting points observed for isolated compounds are 168-170°C (169°C for reference) for scillirosidin, 151-155°C (153°C for reference) for cephalotaxine, 280-288°C (287°C) for stemmadenine, and 168-172°C (169°C for reference) for myxoxanthophyll [37][38][39][40]. e FTIR spectrum of A. candolleanus root extract is shown in Figure 2.
With the help of data and spectrum obtained from the LC-MS-MS technique, phytoconstituents present in the root extract of A. candolleanus have been identified. Four compounds have been isolated and identified using the data of peaks of mass spectral analysis (Figure 3): the terpenoid stemmadenine, the alkaloid cephalotaxine, the glycosides scillirosidin, and myxoxanthophyll ( Figure 4).
Results of LC-MS-MS of A. candolleanus root extract are displayed in Tables 4-7. e LC-MS-MS spectra are given in Figure 3 and           According to earlier studies, terpenoids are found to be more effective against Gram-positive bacteria than Gramnegative bacteria due to their lipophilic properties. Monoterpenes preferentially affect the membrane structures by enhancing the permeability as well as changing the structural arrangement of proteins, producing interference inside the respiratory chain [41]. e natural or synthetic quinolone alkaloids have been found to block the action of topoisomerase enzymes, especially type-II variants, thus preventing nuclear replication [42]. Furthermore, it has been reported that some of the phenols can inhibit the enzymatic activity of the bacterial DNA gyrase by interacting with its ATP site [43]. Concerning the four pure components, stemmadenine, scillirosidin, cephalotaxine, and myxoxanthophyll, isolated from the roots of A. candolleanus, further studies should be conducted on these microorganisms to confirm their effectiveness as well as to elucidate the mechanism of action through which they exert the antibacterial activity. In response to bacterial infection, high levels of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, IL-18, tumor necrosis factor-alpha (TNF-α), and anti-inflammatory cytokine (IL-10) were often found in infected patients. A decrease in IL-6 was associated with a better prognosis instead, and overproduction of IL-10 is considered the main predictor of severity and fatal outcome. In bacterial infections, proinflammatory and anti-inflammatory cytokines are a double-edged sword: on the one hand, they are essential for eradicating the pathogen, but their overproduction can cause tissue and organ damage [44,      e isolated compounds were subjected to STP and PASS analysis to determine the probable biological activities of the substance. Table 8 shows the predictive targets of the bioactive compounds, isolated from A. candolleanus, identified through the STP software. Out of the four compounds tested, we found that scillirosidin and stemmadenine inhibited MAP kinase p38 alpha and beta pathways, responsible for an inflammatory imbalance during bacterial infections, whereas myxoxanthophyll and scillirosidin also target interleukin-8-receptor A and B, which are the major proinflammatory cytokines that get elevated in such patients.
e PASS online predictivity scores for bioactive compounds isolated from the roots of A. candolleanus are reported in Table 9. e possibility that a chemical compound to be active (Pa) or inert (Pi) on a biological target is calculated using the PASS online software. e compounds having a Pa score of greater than 0.7 are considered highly active, while those having a Pa score greater than 0.3 are moderately active. Interestingly, the PASS analysis of the bioactive compounds (stemmadenine, myxoxanthophyll, cephalotaxine, and scillirosidin) revealed that myxoxanthophyll and cephalotaxine were predicted as apoptosis agonists, antioxidants, and anti-inflammatory with a Pa score above 0.7, for each biological activity (Table 9). Taken together, our results highlighted the prominent role of the isolate myxoxanthophyll which was found highly bioactive  Apoptosis agonist Antioxidant Anti-inflammatory Interferon antagonist Cytokine release inhibitor * Pa > 0.7: highly active; Pa > 0.3: moderately active; Pa > 0.1: less active. 8 International Journal of Microbiology and therefore can be considered a promising candidate for drug design studies. From the overall results of drug design, we found that myxoxanthophyll is a promising bioactive isolate with high bioactivity.

Conclusions
e antibacterial activity of A. candolleanus extract was tested against several microbial strains, including B. subtilis, S. aureus, E. coli, S. typhimurium, P. aeruginosa, K. pneumoniae, and K. rhizophila, and results showed a broad inhibition zone against bacterial species. e activity may be a cumulative effect of all the constituents present in the plant. In our study, we have isolated four compounds that were identified as scillirosidin, cephalotaxine, stemmadenine, and myxoxanthophyll using FTIR and LC-MS-MS techniques.
ese compounds will be further tested individually against bacterial strains. We also performed computational studies to predict the most active constituent amongst all four compounds. e results of STP as well as PASS online predictivity score software testing showed that, among the four compounds, myxoxanthophyll was the most active molecule, revealing a predicted activity as an apoptosis agonist, antioxidant, and anti-inflammatory compound, with a Pa score above 0.7. However, this study can be considered a preliminary investigation of the chemical composition and biological activities of A. candolleanus. Further studies are required to validate the activity of these identified compounds using antimicrobial and docking studies.

Data Availability
e data presented in this study are available on request from the corresponding author.

Conflicts of Interest
e authors declare no conflicts of interest.